The dengue viral protease is a heterodimer of nonstructural protein 2B (NS2B) and NS3. The amino-terminal third of NS3 contains four regions of limited sequence homology to trypsin-like serine proteases, including the three active center residues. Two of these four regions in NS3 ("homology boxes" 3 and 4) contain residues predicted to be important for substrate binding (aa 1604, 1605, 1625, 1627 and 1628) based on analogy with the structure of the trypsin P1 specificity pocket. We performed a mutagenesis study of some of the residues in homology boxes 3 and 4 of dengue virus type 2 in order to characterize their requirement for protease activity and to identify mutants with partial cleavage defects. Previously, 46 mutations were analyzed for their effect in vitro on intramolecular cleavage at the NS2B-NS3 junction and intermolecular cleavage at the NS4B-NS5 junction. Subsequently, the crystal structure of the DEN2 NS3 protein was reported by Padmanabhan and Murthy, and the observed effects of most of our mutations were consistent with their expected perturbations of the protease structure. We are now collaborating with these investigators to express, purify, and crystallize various mutant NS3 proteins, to see if the structures are indeed perturbed as predicted. Work is also underway to introduce some of the mutations in NS3 which permit significant residual protease activity back into the dengue genome, in an effort to confirm the observed effect on protease function and possibly to attenuate the virus. Two silent mutations had previously been introduced into the DEN2 infectious clone to create unique Nar I restriction sites which flank the mutagenized region. DEN2 infectious cDNA digested with Nar I is mixed with an Nsi I/BamH I fragment purified from a mutant NS3 clone, and the mixture is used to transform competent yeast to tryptophan independence. In yeast, double homologous recombination between the two DNA fragments results in a full-length DEN2 cDNA clone into which the NS3 mutation of interest has been incorporated. These mutant clones are grown up in E. coli, linearized at the 3' end of the DEN2 genome, and then transcribed into RNA which is electroporated onto monkey kidney cells. In this way, we determine if the construct is still infectious, and isolate mutant viruses from the clones that are. So far, we have made 4 mutant full-length clones: 3 were not infectious (the phenotype of the mutation was lethal), and 1 mutant virus has been produced. The phenotype of this virus will be characterized in growth curves and by a biochemical analysis of protease processing. Also, we will continue to introduce other NS3 mutations back into the infectious clone for characterization.